Targeted human-interferon fusion proteins

ABSTRACT

This disclosure relates to a modified α-helical bundle cytokine, with reduced activity via an α-helical bundle cytokine receptor, wherein the α-helical bundle cytokine is specifically delivered to target cells. Preferably, the α-helical bundle cytokine is a mutant, more preferably it is a mutant interferon, with low affinity to the interferon receptor, wherein the mutant interferon is specifically delivered to target cells. The targeting is realized by fusion of the modified α-helical bundle cytokine to a targeting moiety, preferably an antibody. This disclosure relates further to the use of such targeted modified α-helical bundle cytokine to treat diseases. A preferred embodiment is the use of a targeted mutant interferon, to treat diseases, preferably viral diseases and tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2013/050787, filed Jan. 17, 2013,designating the United States of America and published in English asInternational Patent Publication WO 2013/107791 A1 on Jul. 25, 2013,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial No. 12305075.9, filed Jan.20, 2012.

TECHNICAL FIELD

The disclosure described herein relates to a modified α-helical bundlecytokine, with reduced activity via an α-helical bundle cytokinereceptor, wherein the α-helical bundle cytokine is specificallydelivered to target cells. Preferably, the α-helical bundle cytokine isa mutant, more preferably, it is a mutant interferon, with low affinityto the interferon receptor, wherein the mutant interferon isspecifically delivered to target cells. The targeting is realized byfusion of the modified α-helical bundle cytokine to a targeting moiety,preferably an antibody. This disclosure relates further to the use ofsuch targeted modified α-helical bundle cytokine to treat diseases. Apreferred embodiment is the use of a targeted mutant interferon to treatdiseases, preferably viral diseases and tumors.

BACKGROUND

Cytokines are small proteins that play an important role inintercellular communication. Cytokines can be classified based on theirstructure, the largest group being the four-α-helix bundle family. Thisfamily can, based on the use of receptors, further be divided into theinterferon (IFN) and interleukin (IL)-2, -3, -10 and -12 subfamilies.The α-helical bundle cytokines are important as possiblebiopharmaceuticals for treatment of human diseases. As non-limitingexamples, erythropoietin is used for treatment of anemia or red bloodcell deficiency, somatotropin for treatment of growth hormonedeficiency, and interleukin-2 in the treatment of cancer.

Within the α-helical bundle cytokines, type I IFNs belong to a cytokinefamily having important biological functions. In humans, there are 17different type I IFNs (13α, β, ε, κ, ω), which signal through aubiquitously expressed cell surface receptor composed of two chainsIFNAR1 and IFNAR2. The assembling of the IFN-receptor complex initiatesthe activation of several signal transduction pathways that, dependingupon the cell type, modify cellular differentiation and/or functions.

By acting on virtually every cell type, type I IFN is able to preventproductive viral infection. In addition, it exhibits markedantiangiogenic and proapoptotic effects. Type I IFNs are also deeplyimplicated in the regulation of several functions of the innate andadaptive immunity, as well as on bone homeostasis. It acts particularlyon the activation/differentiation of dendritic cells and osteoclasts.The type I IFN system is, in fact, critically important for the healthof mammals.

Preclinical studies in mice have established a remarkable efficacy oftype I IFN for the treatment of both viral or tumor diseases.Noteworthy, mice cured of an experimental tumor by IFN treatment havebeen found immunized against the initial tumor, suggesting that IFN actsnot only to engage the processes of tumor rejection but also to breakthe immune tolerance against the tumor. Based on these studies, IFNα wasapproved in clinics for the treatment of both viral infection andcancer. More recently, IFNβ was shown to be effective inrelapsing-remitting multiple sclerosis and was also approved for thispathology. Unfortunately, the clinical efficacy of IFN was often founddisappointing and today, other therapeutic strategies such as specificantiviral compounds, chemotherapies and monoclonal antibodies have, whenpossible, largely supplanted IFN broad application. Today, IFN is thefirst line therapeutic choice for only HBV and HCV chronic infectionsand for a limited number of tumors.

The efficacy of type I IFN in clinical practice is limited byineffective dosing due to significant systemic toxicity and sideeffects, including flu-like syndrome, depression, hepatotoxicity,autoimmune disease, thyroid dysfunction and weight loss. It would thusbe highly worthwhile to target IFN activity toward only the cellularpopulation that should be treated with IFN (e.g., infected organ ortumor mass) or activated by IFN (e.g., subsets of immune cells).

In order to solve or limit the systemic toxicity of cytokines, specifictargeting of cytokines by antibody-cytokine fusion proteins has beenproposed (Ortiz-Sanchez et al., 2008). Rossi et al. (2009) specificallydisclose CD20-targeted tetrameric IFNα, and its use in B-cell lymphomatherapy. However, the fusion maintains its biological activity, and iseven more active than commercial pegylated IFN, which means that theunwanted side effects in human treatment would still be present, orwould even be more severe. WO2009039409 discloses targeted IFN and itsapoptotic and anti-tumor activities. Not only does the patentapplication disclose the fusion of an antibody as targeting moiety withwild-type IFN, but also with mutated IFN. However, it is stated that theIFN fragment should retain its endogenous activity at a level of atleast 80%, or even at a higher level than wild-type IFN. Also, in thiscase, the fusion is retaining the unwanted side effects of thewild-type.

SUMMARY OF THE DISCLOSURE

Surprisingly, it was found that a modified α-helical bundle cytokine,with a decreased affinity for the α-helical bundle cytokine receptor anda consequent decreased specific bioactivity, can be fused to a targetingmoiety, wherein the bioactivity is restored toward the targeted cells,but not toward cells that are not targeted by the construct. Suchconstruct has the advantage over the art of having less side effects,especially a lower systemic toxicity, while retaining the bioactivityagainst the target cells.

A first aspect of this disclosure is a targeting construct comprisingmodified α-helical bundle cytokine, characterized by a reduced affinityfor the α-helical bundle cytokine receptor, and a targeting moiety.α-helical bundle cytokines are known to the person skilled in the artand include, but are not limited to, Cardiotrophin-like cytokine NNT-1,Ciliary neurothrophic factor, Macrophage colony stimulating factor,Granulocyte-macrophage colony stimulating factor, Granulocyte colonystimulating factor, Cardiotrophin-1, Erythropoietin, FLT3 ligand,Somatotropin, Interferon α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21,Interferon β, Interferon γ, Interferon κ, Interferon ε, Interferon τ-1,Interferon ω-1, Interleukin 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 α chain, 13,15, 19, 20, 21, 22, 23, 24, 26, 27, 28A, 29, 31, Stem cell factor,Leptin, Leukemia inhibitor factor, Oncostatin M, Prolactin, andThrombopoietin. For a review on α-helical bundle cytokines, see Conklin(2004). A modified α-helical bundle cytokine means that the α-helicalbundle cytokine has been changed to alter the affinity to the receptor,with a final result that the modified α-helical bundle cytokine has areduced affinity for the receptor and a consequent reduced biologicalactivity, as compared to the endogenous wild-type cytokine that bindsnormally to the receptor. Such a modification can be a modification thatdecreases the activity of the normal wild-type cytokine, or it can be amodification that increases the affinity of a homologous, non-endogenousα-helical bundle cytokine (such as, but not limited to, a mouseα-helical bundle cytokine, binding to a human α-helical bundle cytokinereceptor). Modifications can be any modification reducing or increasingthe activity known to the person skilled in the art including, but notlimited to, chemical and/or enzymatic modifications such as pegylationand glycosylation, fusion to other proteins and mutations. Preferably,the modification is a mutation. Even more preferably, it is a mutationdecreasing the affinity of the-α-helical bundle cytokine. A “reducedaffinity” and a “consequent reduced biological activity,” as usedherein, means that the modified α-helical bundle cytokine has abiological activity of less than 70% of the biological activity of theα-helical bundle cytokine; even more preferably, less than 60% of thebiological activity of the α-helical bundle cytokine; more preferably,less than 50% of the biological activity of the α-helical bundlecytokine; more preferably, less than 40% of the biological activity ofthe α-helical bundle cytokine; more preferably, less than 30% of thebiological activity of the α-helical bundle cytokine; more preferably,less than 20% of the biological activity of the α-helical bundlecytokine; and most preferably, less than 10% of the biological activityof the α-helical bundle cytokine as compared to the α-helical bundlecytokine that normally binds to the receptor. Preferably, the modifiedα-helical bundle cytokine is a mutant of the wild-type α-helical bundlecytokine and the activity is compared with the wild type α-helicalbundle cytokine. The affinity and/or the activity can be measured by anymethod known to the person skilled in the art. Preferably, the activityis measured by measuring and quantifying STAT phosphorylation.

A preferred embodiment of the disclosure is a targeting constructcomprising a mutant IFN characterized by reduced affinity for the IFNreceptor and a targeting moiety. IFN can be any IFN including, but notlimited to, IFNα, IFNβ and ω. A “mutant IFN,” as used herein, can be anymutant form that has a lower affinity for the receptor and, as aconsequence, a lower antiproliferative activity and/or a lower antiviralactivity. Indeed, as shown by Piehler et al. (2000), the relativeaffinity correlates directly with the relative antiproliferativeactivity and with the relative antiviral activity. The affinity of themutant IFN to the receptor, in comparison to the affinity of thewild-type IFN to the receptor, can be measured by reflectometricinterference spectroscopy under flow-through conditions, as described byBrecht et al. (1993). The mutant may be a point mutant, a deletion or aninsertion mutant, or a combination thereof. Preferably, the mutant IFNis obtained by active mutagenesis, such as, but not limited to,site-directed mutagenesis by polymerase chain reaction amplification.Preferably, the mutant IFN has a biological activity of less than 70% ofthe biological activity of the wild-type IFN; even more preferably, lessthan 60% of the biological activity of the wild-type IFN; morepreferably, less than 50% of the biological activity of the wild-typeIFN; more preferably, less than 40% of the biological activity of thewild-type IFN; more preferably, less than 30% of the biological activityof the wild-type IFN; more preferably, less than 20% of the biologicalactivity of the wild-type IFN; most preferably, less than 10% of thebiological activity of the wild-type of which it is deduced (i.e., thewild-type IFN of which the coding sequence has been mutated to obtainthe mutant IFN). Mutant forms of IFN are known to the person skilled inthe art. As a non-limiting example, IFNα2 mutants have been listed inPiehler et al. (2000). Preferably, the IFN is a type I IFN. Even morepreferably, the mutant is an IFNα; even more preferably, the mutant isan IFNα2. More preferably, the IFNα2 mutant is mutated in one or moreamino acids of the region 144-154, preferably at positions 148, 149and/or 153; even more preferably, the mutant IFNα2 is selected from thegroup consisting of IFNα2 L153A, IFNα2 R149A and IFNα2 M148A. Mostpreferably, the mutant is selected from the group consisting of IFNα2L153A and IFNα2 R149A.

Preferably, the receptor is IFNAR2.

Preferably, the targeting moiety is targeting to a marker expressed onan IFN receptor-expressing cell, preferably a cell expressing IFNAR2. Inone preferred embodiment, the targeting moiety is directed to atissue-specific marker. Preferably, the tissue is a cancer tissue. Thecancer can be any cancer including, but not limited to, B cell lymphoma,lung cancer, breast cancer, colorectal cancer or prostate cancer. Inanother preferred embodiment, the targeting moiety is directed to amarker selected from the group consisting of Her2 and CD20. In stillanother preferred embodiment, the targeting moiety is directed to a cellsurface marker specific for viral infected cells such as, but notlimited to, influenza M2 protein, LMP1 and EBV proteins). In stillanother embodiment, the targeting moiety is directed toward anosteoclast marker such as DC-STAMP or RANK. Indeed, it is known thatIFN-β plays an important role in bone homeostasis, regulated by RANK andIFNAR coexpressing cells (Abraham et al., 2009). In still anotherembodiment, the targeting moiety is directed toward a markerspecifically expressed on the surface of an immune cell type on whichIFN may regulate activity and/or differentiation. The marker PDL2specifically expressed on dendritic cells and some immune cells is anexample.

A targeting moiety, as used here, can be a protein as a part of aspecifically binding protein complex, or any specifically bindingprotein or protein fragment, known to the person skilled in the art. Itincludes, but is not limited to, carbohydrate binding domains (CBD)(Blake et al., 2006), lectin binding proteins, heavy chain antibodies(hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al.,1994), the variable domain of camelid heavy chain antibodies (VHH), thevariable domain of the new antigen receptors (VNAR), affibodies (Nygrenet al., 2008), alphabodies (WO2010066740), designed ankyrin-repeatdomains (DARPins) (Stumpp et al., 2008), anticalins (Skerra et al.,2008), knottins (Kolmar et al., 2008) and engineered CH2 domains(nanoantibodies; Dimitrov, 2009). Preferably, the targeting moietyconsists of a single polypeptide chain and is not post-translationallymodified. Even more preferably, the targeting moiety is a nanobody.

The targeting construct can be any targeting construct known to theperson skilled in the art. As a non-limiting example, the targetingmoiety may be chemically linked to the mutant interferon, or it may be arecombinant fusion protein. Preferably, the targeting construct is arecombinant fusion protein. The targeting moiety may be fused directlyto the mutant IFN, or it may be fused with the help of a linkerfragment. The targeting moiety may be fused at the aminoterminal or atthe carboxyterminal end of the mutated IFN; preferably, the targetingmoiety is fused at the amino-terminal extremity of the mutated IFNmolecule.

Another aspect of the disclosure is a targeting construct according tothe disclosure for use as a medicament.

Still another aspect of the disclosure is the use of a targetingconstruct according to the disclosure for the manufacture of amedicament to treat cancer.

Still another aspect of the disclosure is the use of a targetingconstruct according to the disclosure for the manufacture of amedicament to treat a viral disease. As a non-limiting example, theviral disease may be HIV infection, HBV infection or HCV infection.

Another aspect of the disclosure is a targeting construct according tothe disclosure for use in treatment of cancer.

Still another aspect of the disclosure is a targeting constructaccording to the disclosure for use in treatment of a viral disease. Asa non-limiting example, the viral disease may be HIV infection, HBVinfection or HCV infection.

Still another aspect of the disclosure is a targeting constructaccording to the disclosure for use in treatment of diseases involvingbone degradation, such as, but not limited to, osteoporosis.

Still another aspect of the disclosure is a pharmaceutical compositioncomprising a targeting construct according to the disclosure and asuitable excipient. It is clear for the person skilled in the art thatsuch a pharmaceutical composition can be used alone, or in a combinationtreatment, such as, but not limited to, a combination with chemotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Representation of the structural elements of the nanobody-IFNfusion protein.

FIG. 2: Firefly luciferase activity induced by the indicated IFNpreparation on HL116 cells (panels A and B) or HL116 cells expressingthe murine leptin receptor (mLR) (panels C and D). Panels A and C on theone hand, and panels B and D on the other hand, were generated in twoseparate experiments. Consequently, only vertical comparison (panel Aversus panel C or panel B versus panel D) is possible.

FIG. 3: Renilla (light grey) and Firefly (dark grey) luciferase activityinduced by the nanobody-IFNα2R149A or by the IFNα2 (7 pM) in a 1:1coculture of cells expressing the leptin receptor and an IFN-induciblefirefly luciferase or in cells expressing an IFN-inducible renillaluciferase but devoid of leptin receptor. Luciferase activities areexpressed as a percentage of the luciferase activities induced by 3 nMIFN α2.

FIGS. 4A and 4B: Activity of the purified constructs targeting the mLR:FIG. 4A, Quantification of their specific activities on cells expressingthe target (HL116-mLR) or on cells lacking the target (HL116). FIG. 4B,Calculation of the targeting efficiencies of the different constructs.

FIG. 5: Activity of the construct 4-11-IFNA2-R149A in presence andabsence of the unconjugated leptin receptor binding nanobody. HL116cells expressing the mLR were incubated for 6 hours with either theIFN-α2 (IFNA2) or the IFNA2-R149A fused to the nanobody 4-11(Nanobody-IFNA2-R149A) at their respective EC50 concentration in thepresence or absence (control) of a 100-fold molar excess of free 4-11nanobody.

FIG. 6: Targeting the mutant IFN using the leptin binding nanobody 4-10.

FIG. 7: Firefly luciferase activity induced in HL116 cells expressingthe mLR by the nanobody-IFNα2R149A in the presence of anti IFNAR1monoclonal antibody 64G12 (Benoit et al., J. Immunol. 150:707-716, 1993)or anti IFNAR2 monoclonal antibody MMHAR2 (PBL Interferon Source).

FIGS. 8A and 8B: Specificity of the targeting of 4-11-IFNA2-R149A tocells expressing the mLR. FIG. 8A, Cytopathic effect of the EMCV onHL116 cells (dark gray symbols) or on HL116-mLR (light grey symbols) ofparental IFNA2 (upper left panel) or of the 4-11-IFNA2-R149A (lower leftpanel). FIG. 8B, Upper panel: calculated EC50 for antiviral activity;lower panel: calculated targeting efficiencies.

FIGS. 9A and 9B: Specific activities (EC50) of IFNα2 (FIG. 9A) and thenanobody-IFNα2R149A (2R5A; FIG. 9B) on BXPC3 and BT474 cell lines, whichexpress different number of Her2 molecule at their surface (10.9×10³ and478×10³, respectively). The ordinate scale of FIG. 9A cannot be comparedto the ordinate scale of FIG. 9B.

FIG. 10: Targeting of the 1R59B-IFNA2-Q124R to human Her2 expressingmouse cells. Quantification of the OASL2 mRNA expression in BTG9A cellswith and without Her2 expression.

FIG. 11: Targeting of mutant IFNA2 to human Her2 expressing mouse cells,using a single chain antibody. Quantification of the ISG15 mRNAexpression in BTG9A cells with and without Her2 expression.

FIG. 12: Control of the activation of Her2 phosphorylation: Lanes 13 to76: no phosphorylated Her2 in extract of BTG9A cells expressing humanHer2 treated with different concentration (200 pM for lanes 3 to 5, 2 nMfor lane 6) and time (lane 3: 5 minutes, lanes 4 and 6: 10 minutes, lane5: 30 minutes) with the construct 1R59B-IFNA2-Q124R. Lanes 7 and 8:control for the detection of phosphorylated Her2 in the human BT474 cellline. Lane 1: extract of BTG9A cells. Lane 2: extract of BTG9A cellsexpressing human Her2.

FIG. 13: Targeting the anti-PD-L2 122-IFNA2-Q124R to mouse primary cellsendogenously expressing PD-L2. The activation is measured as STATphosphorylation. The light gray area represents the PD-L2-negative cellpopulation; the dark gray area represents the PD-L2-positive population.

FIG. 14: In vivo targeting of 122-IFNA2-Q124R to PD-L2 expressing cells.Mice were injected intraperitoneally (IP) or intravenous (IV) witheither PBS, a control construct (nanobody against GFP fused to mutantIFNA2-Q124R, indicated as control) or a targeted mutant IFN (targeted toPD-L2, Nb122-IFN2-Q124R, indicated as 122-Q124R. The light gray arearepresents the PD-L2-negative cell population; the dark gray arearepresents the PD-L2-positive population.

FIG. 15: Dose response curve after IV injection of 122-IFN-Q124R inmice. The light gray area represents the PD-L2-negative cell population;the dark gray area represents the PD-L2-positive population.

FIG. 16: Leptin-dependent growth induced by targeted mutant leptin: theloss in activity of a mutant leptin can be rescued in Ba/F3 cellsexpressing the human TNFR1. First two panels, experiment using theH6-leptin construct; second two panels, experiment using the mleptinconstruct. H6 indicated the his tag (6×his).

FIG. 17: construction of the targeted leptin constructs (SEQ ID NOS:9and 10).

DETAILED DESCRIPTION Examples

Materials and Methods to the Examples

Nanobodies and ScFv

The nanobody 4-11 directed against the murine leptin receptor wasdescribed in Zabeau et al. (2012), and in the patent application WO2006/053883. Its coding sequence is cloned into the mammalian expressionvector pMET7 (Takebe et al., 1988) in fusion with the SIgk leaderpeptide, the HA tag and albumin. Plasmid name: pMET7SIgK-HA-4.11-Albumin.

The nanobody 4-10 is also described in Zabeau et al. (2012).

The anti Her2 nanobodies 1R59B and 2R5A are described in Vaneycken etal. (2011). They were fused to the human IFNA2-Q124R and to the humanIFNA2-R149A in the pMET7 vector. Fusion protein was produced bytransfection of 293T cells.

The anti PD-L2 nanobody 122 was from Johan Grooten (VIB, Gent, Belgium).It was fused to the human IFNA2-Q124R in the pMET7 vector. The fusionprotein was produced by transfection of 293T cells and purified usingthe HisPur Ni-NTA purification kit (Pierce, Thermo Scientific).

The anti TNF nanobody was obtained from Claude Libert (VIB).

The anti Her2 ScFv was obtained from Andrea Plückthun (Wörn et al.,1998). It was fused to the human IFNA2-Q124R in the pMET7 vector. Thefusion protein was produced by transfection of 293T cells.

Control nanobody against GFP was obtained from Katrien Van Impe(University Ghent).

Interferons

The IFNα2 and the mutants L153A and R149A, which show an IFNAR2 affinityreduced by a factor 10 and 100, respectively, have been described inRoisman et al. (2001). IFN coding sequences are cloned in the pT3T7vector (Stratagene) in fusion with the ybbR tag. Plasmid names:pT7T3ybbR-IFNa2, pT7T3ybbR-IFNa2-L153A, pT7T3ybbR-IFNa2-R149A.

The human IFNA2 Q124R has a high affinity for the murine IFNAR1 chainand a low affinity for the murine IFNAR2 chain. (Weber et al., 1987.)

Nanobody-IFN Fusion Construction

The coding sequence of the IFNα2, wild-type, L153A and R149A weresynthesized by PCR from the corresponding pT3T7ybbR IFNa2 plasmids usingthe Expand High Fidelity PCR system from Roche Diagnostics and thefollowing primers: Forward:5′GGGGGGTCCGGACCATCACCATCACCATCACCATCACCATCACCCTGCTTCTCCCGCCTCCCCAGCATCACCTGCCAGCCCAGCAAGTGATAGCCTGGAATTTATTGC3′ (SEQ ID NO: 1),Reverse: 5′ CGTCTAG ATCATTC CTTACTTCTTAAAC3′ (SEQ ID NO: 2). This PCRintroduces a His tag and a series of five Proline-Alanine-Serine (PAS)repeats at the amino terminal extremity of the IFNs. The PCR productswere digested with BspEI and XbaI and cloned into BspEI-XbaI digestedpMET7 SIgK-HA-4.11-Albumin vector to obtain pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNA2-L153A and pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNA2-R149A.

In a similar way, the human mutant Q124R was fused to the 1R59B nanobodyand to the anti-PD-L2 nanobody.

Production of the Nanobody-IFN Fusion Protein

HEK293T cells were grown in DMEM supplemented with 10% FCS. They weretransfected with pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNA2-L153A pMET7SIgK-HA-4.11-His-PAS-ybbr-IFNA2-R149A, pMET7SIgK-HA-2R5A-His-PAS-ybbr-IFNA2-R149A, pMET7SIgK-HA-1R59B-His-PAS-ybbr-IFNA2-Q124R, pMET7SIgK-HA-4D5-His-PAS-ybbr-IFNA2-Q124R or pMET7SIgK-HA-122-His-PAS-ybbr-IFNA2-Q124R using lipofectamin (Invitrogen). 48hours after the transfection, culture mediums were harvested and storedat −20° C.

Alternatively, sequences encoding the different nanobody-IFN fusionswere subcloned into the baculovirus transfer plasmid pBAC-3 (Novagen).Proteins were produced by insect cells using the BacVector kit (Novagen)and purified to homogeneity using the HisPur Ni-NTA purification kit(Pierce, Thermo Scientific) and gel filtration. Protein concentrationswere measured by absorbance at 280 nm.

IFN Reporter Cell Lines

The HL116 clone (Uzê et al., 1994) is derived from the human HT1080 cellline. It contains the firefly luciferase gene controlled by theIFN-inducible 6-16 promoter. The HL116 cells were co-transfected with anexpression vector encoding the short isoform of the murine leptinreceptor (pMET7 mLRsh-FLAG, Eyckerman et al., 1999) and pSV2neo(Southern and Berg 1982). Stable transfected clones were isolated inG418-containing medium. The clone 10 was selected after analysis of thesurface expression level of the murine leptin receptor by FACS, usingthe biotinylated anti-mouse leptin receptor antibody BAF497 from R&D andstreptavidin-APC (BD Bioscience).

HT1080 cells were cotransfected with p6-16-RL, a plasmid encoding theRenilla luciferase (from pRL-null, Promega) controlled by theIFN-inducible 6-16 promoter (from p1.8gpt-5, Pellegrini et al., 1989),pBB3 (Bourachot et al., 1982) and salmon sperm DNA (Sigma). Stabletransfected clones were isolated in HAT-containing medium. The clone 4was selected for a high level of renilla luciferase activity inductionupon IFN induction.

The human pancreatic carcinoma BXPC3 (Tan et al., 1986; ATCC: CRL 1687)and breast cancer BT474 (Lasfargues et al., 1979; ATCC: HTB-20) celllines were obtained from ATCC.

The mouse BTG9A cells were described in Uzê et al. (1990).

Measurement of the Luciferase Activities

IFN-specific activities were measured by quantifying the luciferaseactivity induced in HL116 cells and on the HL116 clone 10 expressing themLR. The EC50 were calculated using non-linear data regression withGraphPad Prism software.

Luciferase activities were determined on a Berthold centro LB960luminometer using either the Firefly Luciferase Assay System or theDual-Luciferase Reporter Assay System from Promega after six hours IFNstimulation.

Quantitative RT-PCR

The expression of the interferon inducible gene 6-16 was quantified byRT-PCR relative to GAPDH or β-actin. Cells were treated with targeted orcontrol IFN for 4 hours. Total RNA was purified with RNEASY® columns(Qiagen). Reverse transcriptions were primed with random primers andperformed using Moloney murine leukemia virus reverse transcriptase(Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using aLIGHTCYCLER® as described (Coccia et al., 2004).

For Her2, the transfection culture medium was assayed on murine BTG9Aand BTG9A cells expressing human Her2 for expression of the OASL2 generelatively to the expression of the β actin gene by quantitative RT-PCRusing a Light Cycler (Roche) and the following primers: OASL2 forward:CAC-GAC-TGT-AGG-CCC-CAG-CGA (SEQ ID NO: 3); OASL2 reverse:AGC-AGC-TGT-CTC-TCC-CCT-CCG (SEQ ID NO: 4); βactin forward:AGA-GGG-AAA-TCG-TGC-GTG-AC (SEQ ID NO: 5); βactin reverse:CAA-TAG-TGA-TGA-CCT-GGC-CGT (SEQ ID NO: 6). In a similar way the ISGexpression in Her2 targeted cells was measured using the same βactinprimers and the following ISG15 primers: ISG15 forward:GAG-CTA-GAG-CCT-GCA-GCA-AT (SEQ ID NO: 7); ISG15 reverse:TTC-TGG-GCA-ATC-TGC-TTC-TT (SEQ ID NO: 8).

Antiviral Assay

The antiviral assay was performed using the EMC virus and scoring thevirus replication-dependent cytopathic effect as described in Stewart(1979).

Measurement of her2 Phosphorylation

BTG9A cells expressing human Her2 were treated with 200 pM to 2 nM of 1R59B-IFNA2-Q124R for 10 to 30 min. Cells were lysed in RIPA, andanalysed by western blot on an ODYSSEY FC (gels imaging system usingvisible, near-infrared or chemiluminesce signals, Licor Bioscience)after 7% SDS-PAGE (40 μg lane). Phopho-Her2 was detected with the antiHer2 Y-P 1248 (Upstate #06-229) and the Goat anti rabbit secondaryantibody IRDye 680 (Licor Bioscience #926-32221).

Measurement of STAT1 Phosphorylation

STAT1 phosporylated on Y701 were detected by FACS using the STAT1-PY701(PE) (Beckton Dickinston #612564) and the manufacturer instruction forthe PHOSFLOW (flow cytometry-based protein phosphorylation detection)technology.

Targeted Leptin Constructs

The sequence of the targeted leptin constructs is given in FIG. 17. TheL86 that is indicated is the amino acid that is mutated either to S orN.

Example 1 The Nanobody-Interferon Fusion Proteins

FIG. 1 shows a schematic representation of the nanobody-IFN fusionproteins constructed with either IFNα2 wild-type or the L153A and R149Amutants.

Example 2 IFN Activity of the Nanobody-IFN Fusion Proteins is TargetedToward Murine Leptin Receptor Expressing Cells

The three nanobody fusion proteins with IFNα2 WT, IFNα2 L153A or R149Awere assayed on both HL116 and HL116-mLR-clone 10 cells, which expressthe murine leptin receptor. The IFNα2 alone was also assayed in thisassay system in order to check that the two cell clones do not differ intheir IFN responsiveness. Indeed, both HL116 and HL116-mLR-clone 10cells are equally sensitive to this IFN (FIGS. 2A and 2C, blacksymbols). The IFN activity of the three nanobody-IFN fusion proteins is,however, dramatically increased in cells expressing the leptin receptorcompared to parental HL116 cells (compare FIG. 2A with FIG. 2C and FIG.2B with FIG. 2D).

It was estimated that cells expressing the leptin receptor are 10-, 100-and 1000-fold more sensitive than parental HL116 cells to thenanobody-IFN WT, L153A and R149A, respectively. Since the affinities forIFNAR2 of the IFN mutant L153A and R149A are 0.1 and 0.01 relative tothe WT, there is a correlation between the loss of activity caused bymutations in the IFNAR2 binding site and the targeting efficiency by thenanobody.

In order to determine whether the IFN activity of the nanobody-IFNfusion proteins is delivered only on cells expressing the nanobodytarget or also on neighboring cells, the nanobody-IFNα2R149A was assayedon a coculture of HL116-mLR-clone10 and HT1080-6-16 renilla luciferaseclone4. Both cell types will express luciferase activity in response toIFN stimulation, but cells expressing the target of the nanobody willdisplay a firefly luciferase activity, whereas cells devoid of leptinreceptor will display a renilla luciferase activity. The dilution of thenanobody-IFNα2R149A protein was chosen at 1/30, a dilution that inducesa maximal response in cells carrying the leptin receptor and a minimalresponse on cells devoid of the nanobody target (see FIGS. 2B and 2D,black curves). FIG. 3 shows clearly that the renilla luciferase activityis not induced upon stimulation of the co-culture with thenanobody-IFNα2R149A, indicating that the targeted IFN activity isdelivered only on cells expressing the antigen recognized by thenanobody.

The efficacy of the targeting is further illustrated by comparing theactivity of wild-type and two types of mutant IFN (L153A and R149A) whenadded to HL116 expressing or not expressing the murine leptin receptorthat is used for the targeting. The results clearly show that theactivity of the mutants is higher when the construct is targeted, andthat the effect of targeting for the mutant is bigger than for wild-type(FIGS. 4A and 4B).

In order to prove that the targeting was nanobody specific, HL116 cellsexpressing the mLR were incubated for 6 hours with either the IFN-α2(indicated as IFNA2) or the IFNA2-R149A fused to the nanobody 4-11(Nanobody-IFNA2-R149A) at their respective EC50 concentration in thepresence or absence (control) of a 100-fold molar excess of free 4-11nanobody. Cells were lysed and the IFN-induced luciferase activitieswere measured. As shown in FIG. 5, the non-targeted IFN is not inhibitedby the free nanobody, while the targeted construct is stronglyinhibited, showing the specific effect of the targeting.

The targeting to the leptin receptor is independent of the epitope onthe receptor: using the anti-leptin receptor nanobody 4-10 (Zabeau etal., 2012), which recognizes a different domain on the receptor than thenanobody 4-11, a similar activation can be obtained using a targetedmutant IFN (FIG. 6).

Example 3 The IFN Activity of the Nanobody-IFN Fusion Proteins on CellsExpressing the Leptin Receptor is Mediated by Both IFN Receptor Chains

In order to determine whether the IFN activity of the nanobody-IFNfusion proteins needs the activation of the IFN receptor, HL116 cellsexpressing the murine leptin receptor were pretreated with neutralizingantibodies against IFNAR1 or IFNAR2, and then stimulated with thenanobody-IFNA2-R149A fusion protein. The activity of the IFN-inducedluciferase was measured. FIG. 7 shows that both anti-IFNAR1 andanti-IFNAR2 neutralizing antibodies inhibit the IFN activity of thenanobody-IFNA2-R149A.

Example 4 Target-Specific Induction of Antiviral Activity by4-11-IFNA2-R149A in Cells Expressing the Murine Leptin Receptor

Antiviral activity is an integrated part of the IFN response, implyingthe expression of several genes. Therefore, the antiviral activity onmLR-expressing cells was controlled, after targeting the mutant R149AIFN using the anti-leptin receptor antibody 4-11. The results aresummarized in FIG. 8. The activity was measured as the cytopathic effecton HL116 cells, with or without leptin receptor expression. The specificantiviral activity of the 4-11-IFNA2-R149A nanobody-IFN fusion proteinis 716-fold higher when assayed on leptin receptor-expressing cellscompared to HL116 cells.

Example 5 Targeting of IFN Activity on Her2 Expressing Cells

In order to demonstrate that the concept is not restricted to cytokinereceptor targeting, we generated similar fusion protein using thenanobody 2R5A against Her2 (Vaneycken et al., 2011) and the mutant IFNalpha2 R149A (2R5A-IFNA2-R149A). This molecule was assayed on BXPC3(Pancreatic cancer, from ATCC) and BT474 (Breast cancer, from ATCC) celllines and compared with the activity of IFN-α2 (IFNA2) for the inductionof the 6-16 IFN-inducible gene as determined relative to GAPDH byquantitative RT-PCR. The BXPC3 and BT474 cells lines differ by theirnumber of Her2 molecules expressed at their surface (10.9×10³ and478×10³, respectively as reported by Gaborit et al. (2011)).

FIGS. 9A and 9B show the EC₅₀ determination of IFNA2 activity (FIG. 9A)and 2R5A-IFNA2-R149A activity (FIG. 9B) for the induction of theIFN-inducible gene 6-16 on BXPC3 and BT474 cell lines. FIG. 9A showsthat BXPC3 and BT474 cell lines exhibit the same sensitivity to IFN-α2.FIG. 9B shows that the 2R5A-IFNA2-R149A nanobody-IFN fusion protein ismuch more potent on the BT474 cell line, which expresses 40-fold moreHer2 molecule than BXPC3.

In conclusion, the concept that consists of targeting type I IFNactivity on cells expressing a specific cell surface antigen, as shownon human cells expressing the mouse leptin receptor, can be extended tountransfected human cells expressing another cell surface molecule froma different structural family, at a level naturally found in severaltypes of breast carcinoma.

Example 6 Targeting of Mutant IFNA2-Q149R to Mouse Cells ExpressingHuman Her2

Mutant human IFNA2 Q149R was targeted to murine cells, expressing thehuman Her2, using the nanobody 1R59B in the 1R59B-IFNA2-Q124R. The IFNA2Q124R has a high affinity for the murine IFNAR1 chain and a low activityfor the murine IFNAR2 chain (Weber et al., 1987). The induction by IFNwas measured as expression of the OASL2 messenger RNA, by RT-QPCR. Theresults are shown in FIG. 10. There is clearly a targeting-specificinduction in the Her2-expressing cells, whereas there is no significantexpression detected in untransfected BTG9A cells.

Similar results were obtained when the Her2-specific ScFv against Her2was used to target the mutant IFN Q124R. In this case, the IFN inductionwas measured using the ISG15 messenger RNA expression. The results areshown in FIG. 11. Again, a specific induction of ISG15 is seen in thecells expressing Her2, while there is little effect of the mutant IFN onthe cells that do not express Her2.

Example 7 The Construct 1R59B-IFNA2-Q124R Does Not Activate thePhosphorylation of Her2

To check whether targeting of Her2 is resulting in Her2 activation, Her2phosphorylation was controlled in targeted cells. The results are shownin FIG. 12, clearly demonstrating that no phosphorylated Her2 could bedetected in 1R59B-IFNA2-Q124R targeted cells, irrespective of theconcentration or time of treatment.

Example 8 The Anti PD-L2 Nb122-IFNA2-Q124R Construct Activity isTargeted on Mouse Primary Cells Expressing PD-L2

Cells from a mouse peritoneal cavity were isolated and treated in vitrowith Nb122-IFNA2-Q124R or natural mIFNα/β for 30 minutes. Cells were,fixed, permeabilized, labelled with antibodies against PD-L2 (APC) (BD#560086) and STAT1-PY701 (PE) (BD #612564) and analyzed by FACS.

The PD-L2-positive cell population represents 20% of the total cellpopulation present in the mouse peritoneal cavity.

The results are shown in FIG. 13. It is clear from this figure that inuntreated cells, or in non-targeted, murine IFN-treated cells, the peaksof STAT1-P for PD-L2 expressing and non-expressing cells coincide.Moreover, a clear induction in STAT1-P can be seen by murine IFNtreatment. Treatment with the targeted mutant IFN, however, results in aspecific shift in the STAT1-P only for the PD-L2 expressing cells.

The same result is obtained if the IFN response of splenocytes isanalyzed in a similar experiment. The PD-L2-positive cell populationrepresents 1% of the total cell population present in mouse spleen,indicating that also a minor cell population can be targeted in anefficient way.

Example 9 In Vivo Injection of 122-IFNA2-Q124R Construct Induces an IFNResponse Only in PD-L2-Expressing Cells

Mice were injected (IP or IV) with either PBS, Nb122-IFNA2-Q124R or acontrol Nb (against GFP) fused to IFNA2-Q124R. 30 min post injection,mice were killed, cells from the peritoneal cavity were recovered bywashing the peritoneal cavity with PBS, fixed (PHOSFLOW (flowcytometry-based protein phosphorylation detection) Fix buffer I BD#557870), permeabilized (PHOSFLOW (flow cytometry-based proteinphosphorylation detection) Perm buffer III, BD #558050), labelled withAbs against PD-L2 (APC) (BD #560086) and STAT1-PY701 (PE) (BD #612564)and analysed by FACS. The results are shown in FIG. 14. STAT1-Pcoincides in PD-L2 positive and negative cells treated with either PBSor control nanobody. However, a clear induction in STAT1-P (only in thePDL2 positive cell population) can be seen when the mice are injectedwith the targeted mutant IFN.

As a control, STAT1-P was checked in mice, iv injected with differentdoses of natural mouse IFN (10,000, 100,000 or 1,000,000 units), and nodifference in STAT1-P could be detected between the PD-L2-positive andPD-L2-negative cells.

FIG. 15 shows a similar dose response curve after iv injection of theNb122-IFNA2-Q124R construct. A shift in STAT1-P in the PD-L2 expressingcells can be noticed even at the lowest dose of 64 ng.

Example 10 Targeting of Mutant Leptin to the Leptin Receptor, Using aTruncated TNFα Receptor

Ba/F3 cells are growth-dependent on IL-3. After transfection with themLR, Ba/F3 cells also proliferate with leptin. Leptin mutants withreduced affinity for their receptor are less potent in inducing andsustaining proliferation of Ba/F3-mLR cells. Leptin mutant L86S has amoderate, and mutant L86N has a strong, reduction in affinity and,hence, a moderate and strong reduced capacity to induce proliferation,respectively.

Additional transfection of Ba/F3-mLR cells with the human TNFα Receptor1 (hTNFR1) lacking its intracellular domain introduces a non-functionalreceptor, which can function as a membrane-bound extracellular marker.

Chimeric proteins consisting of leptin and a nanobody against humanTNFR1 (here nb96) will bind to cells carrying the mLR and to cellscarrying the hTNFR1. Chimeric proteins with leptin mutants L86S and L86Nhave reduced affinity for the LR but retain their affinity for thehTNFR1.

Chimeric proteins were produced by transient transfection of Hek293Tcells with expression plasmids. Supernatant was 0.45 μm filtered andserially diluted in 96-well plates for the assay. A serial dilution ofpurified recombinant leptin was used as a reference. 3000 to 10000 cellswere plated per well and proliferation was measured by staining with XTTfour or five days later. OD was measured at 450 nm. The results areshown in FIG. 16, for two experiments using a different leptin construct(see FIG. 17). For both constructs, an hTNFR depending growthstimulation can be seen for the mutant constructs, whereas the hTNFRexpression does not affect the growth of the cells treated with wt(non-targeted) leptin. It is clear from these results that the targetingcan compensate for the negative effect of the mutation.

REFERENCES

-   Abraham, A. K., M. Ramanathan, B. Weinstock-Guttman, and D. E. Mager    (2009). Biochemical Pharmacology 77:1757-1762.-   Benoit, P., D. Maguire, I. Plavec, H. Kocher, M. Tovey, and F. Meyer    (1993). J. Immunol. 150:707-716.-   Blake, A. W., L. McCartney, J. Flint, D. N. Bolam, A. B.    Boraston, H. J. Gilbert, and J. P. Knox (2006). J. Biol. Chem.    281:29321-29329.-   Bourachot, B., J. Jouanneau, I. Giri, M. Katinka, S. Cereghini,    and M. Yaniv (1982). EMBO J. 1:895-900.-   Brecht, A., G. Gauglitz, and J. Polster (1993). Biosens.    Bioelectron. 8:387-392.-   Coccia, E. M., M. Severa, E. Giacomini, D. Monneron, M.-E.    Remoli, I. Julkunen, M. Cella, R. Lande, and G. Uzé (2004). Eur. J.    Immunol. 34:796-805.-   Conklin, D. (2004). J. Computational Biol. 11:1189-1200.-   Dimitrov, D. S. (2009). mAbs 1:26-28.-   Eyckerman, S., W. Waelput, A. Verhee, D. Broekaert, J.    Vendekerckhove, and J. Tavernier (1999). Eur. Cytok. Netw.    10:549-559.-   Gaborit, N., C. Labouret, J. Vallaghe, F. Peyrusson, C.    Bascoul-Mollevi, E. Crapez, D. Azria, T. Chardès, M. A. Poul, G.    Mathis, H. Bazin, and A. Pélegrin (2011). J. Biol. Chem.    286:11337-11345.-   Ortiz-Sanchez, E., G. Helguera, T. R. Daniels, and M. L. Penichet    (2008). Expert Opin. Biol. Ther. 8:609-632.-   Pellegrini, S., J. John, M. Shearer, I. M. Kerr, and G. R. Stark    (1989). Mol. Cell. Biol. 9:4605-4012.-   Piehler, J., L. C. Roisman, and G. Schreiber (2000). J. Biol. Chem.    275:40425-40433.-   Lasfargues, E. Y., W. G. Coutino, and A. S. Dion (1979). In Vitro    15:723-729.-   Nygren, P. A. (2008). FEBS J. 275:2668-2676.-   Roisman, L. C., J. Piehler, J. Y. Trosset, H. A. Scheraga, and G.    Schreiber (2001). Proc. Natl. Acad. Sci. USA 98:13231-13236.-   Rossi, E. A., D. M. Goldenberg, T. M. Cardillo, R, Stein and C. H.    Chang (2009) Blood 114:3964-3871.-   Southern, P. J. and P. Berg (1982). J. Mol. Appl. Genet. 1:327-341.-   Skerra, A. (2008). FEBS J. 275:2677-2683.-   Stewart II W. E. The Interferon System, Springer-Verlag, Wien, N.Y.    1979.-   Stumpp, M. T., H. K. Binz, and P. Amstutz (2008). Drug Discov. Today    13:695-701.-   Takabe, Y., M. Seiki, J. Fujisawa, P. Hoy, K. Yokata, and N. Arai    (1988). Mol. Cell. Biol. 8:466-472.-   Tan, M. H., N. J. Nowak, R. Loor, H. Ochi, A. A. Sandberg, C.    Lopez, J. W. Pickren, R. Berjian, H. O. Douglass, Jr., and T. M. Chu    (1986). Cancer Invest. 4:15-23.-   Tramontano, A., E. Bianchi, S. Venturini, F. Martin, A. Pessi,    and M. Sollazzo (1994). J. Mol. Recognition 7:9-24.-   Uzê et al. (1990). Cell 60:225-234.-   Uzê, G., S. Di Marco, E. Mouchel-Veilh, D. Monneron, M. T.    Bandu, M. A. Horisberger, A. Dorques, G. Lutfalla, and K. E.    Mogensen (1994). J. Mol. Biol. 243:245-257.-   Vaneycken, I., N. Devoogdt, N. Van Gassen, C. Vincke, C. Xavier, U.    Wernery, S. Muyldermans, T. Lahoutte, and V. Caveliers (2011).    FASEB J. 25:2433-2446.-   Weber et al. (1987). EMBO J. 6:591-598.-   Wörn et al. (1998). FEBS Lett. 427:357-361.-   Zabeau, L., A. Verhee, D. Catteeuw, L. Faes, S. Seeuws, T.    Decruy, D. Elewaut, F. Peelman, and J. Tavernier (2012). Biochem. J.    441:425-434.

The invention claimed is:
 1. A composition comprising a targetingconstruct, the targeting construct comprising: mutated human interferonalpha 2, said mutated human interferon alpha 2 having a R149A mutationand a reduced affinity as compared to the wild type human interferonalpha 2, and a targeting moiety, said targeting moiety comprises anantibody directed to a programmed death-ligand 2 (PD-L2), wherein thetargeting moiety restores the reduced affinity of the mutated humaninterferon alpha 2 for IFNAR2 on targeted cells.
 2. The targetingconstruct according to claim 1, wherein said antibody is a nanobody. 3.A pharmaceutical composition comprising a targeting construct, thetargeting construct comprising: the composition according of claim 1;and a suitable excipient.
 4. The composition of claim 1, furthercomprising a linker wherein the mutated human interferon alpha 2 and thetargeting moiety are connected via a linker.